Buffering action of endogenous nitric oxide on the adrenocortical secretagogue effect of endothelins in the rat

The secretagogue effect of endothelins (ETs) on the rat adrenal cortex is mediated by the ETB receptor. ETB receptors are coupled with nitric oxide (NO) synthase (NOS), and NO is known to inhibit steroid-hormone secretion from adrenal cortex. We investigated whether ETB-mediated NO production interferes with the stimulatory action of ETs on rat adrenal cortex. The selective agonist of ETB receptor BQ-3020 concentration-dependently increased aldosterone secretion from dispersed zona glomerulosa (ZG) cells and corticosterone secretion from dispersed zona fasciculata-reticularis (ZF/R) cells, and the NOS inhibitor NG-nitro-L-arginine methylester (L-NAME) potentiated the effect of BQ-3020 in a concentration-dependent manner. The guanylate cyclase inhibitor Ly-83583, at a concentration suppressing guanylin- and L-arginine-induced cyclic-GMP release from dispersed adrenocortical cells, did not affect the secretory response of ZG and ZF/R cells to BQ-3020. ET-1, an agonist of both ETA and ETB receptors, stimulated the release of both aldosterone and corticosterone by in situ perfused rat adrenal gland. This effect was potentiated by L-NAME and unaffected by Ly-83583. Collectively, our findings allow us to suggest that endogenous NO exerts in vivo and in vitro a cyclic-GMP-independent buffering action on the ETB receptor-mediated adrenocortical secretagogue action of ETs.     

Signaling pathways involved in the A and B receptor-mediated cortisol secretagogue effect of endothelins in the human adrenal cortex

Endothelins (ETs) are a family of 21-amino acid hypertensive peptides, which together with their receptors ETA and ETB are expressed in human adrenal cortex. Evidence has been provided that ETs exert a potent secretagogue effect on human adrenocortical cells, acting through both ETA and ETB receptors. Therefore, it seemed worthwhile to study the signaling cascades mediating the cortisol secretagogue effect of the two receptor subtypes. Normal adrenal glands were obtained from consenting patients undergoing unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. Dispersed zona fasciculata-reticularis (ZF/R) cells were obtained by collagenase digestion and mechanical disaggregation. The selective activation of ETA and ETB receptors was obtained by exposing dispersed cells to ET-1 plus the ETB receptor antagonist BQ-788 and to the selective ETB receptor agonist BQ-3020, respectively. ETA and ETB receptors about equally contributed to the cortisol response of dispersed ZF/R cells to ETs. The phospholipase (PL) C inhibitor U-73122 abolished ETA-mediated secretory response, but only partially prevented the ETB-mediated one. The phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase (PK) C inhibitor calphostin-C significantly blunted the secretory responses ensuing from the activation of both receptor subtypes, while the Ca(2+)-channel blocker nifedipine was ineffective. The ETB receptor-, but not the ETA receptor-mediated cortisol response was partially reversed by the cyclooxygenase (COX) inhibitor indomethacin, which when added together with U-73122 abolished it. The inhibitors of adenylate cyclase, PKA, tyrosine kinase and lipoxygenase did not affect the secretory response to the activation of either receptor subtype. ETA-receptor activation raised inositol triphosphate (IP3) production from dispersed ZF/R cells, while ETB-receptor stimulation enhanced both IP3 and prostaglandin-E(2) production. Collectively, our findings indicate that ETs stimulate cortisol secretion from human ZF/R cells, acting through ETA receptors exclusively coupled with PLC/PKC-dependent pathway and ETB receptors coupled with both PLC/PKC- and COX-dependent cascades.     

Mechanisms and receptor subtypes involved in the stimulatory action of endothelin-1 on rat adrenal zona glomerulosa

Endothelin (ET)-1 is the prototype of a family of 21-amino acid residue hypertensive peptides, acting through two subtypes of receptors, named ETA and ETB. ETs and their receptors are expressed in the adrenal cortex and medulla, and ET-1 enhances both corticosteroid and catecholamine release. ET-1 concentration-dependently (from 10(-11) to 10(-8) M) increased aldosterone secretion of both dispersed rat zona glomerulosa (ZG) cells and adrenal slices containing a core of medullary chromaffin tissue, but the response of the latter preparations was significantly more intense than that of the formers. The stimulatory effect of 10(-8) M ET-1 on dispersed ZG cells was blocked by the ETB-receptor antagonist BQ-788 (10(-7) M), but not by the ETA-receptor antagonist BQ-123 (10(-7) M); conversely, both ET-receptors antagonists counteracted aldosterone response of adrenal slices to ET-1. The -adrenoceptor antagonist l-alprenolol (10(-6) M) did not affect aldosterone response of dispersed ZG cells to ET-1 (10(-8) M), but it significantly lowered that of adrenal slices. l-Alprenolol also counteracted the aldosterone response of adrenal slices to the pure activation of ETB or ETA receptors, as obtained by using the selective ETB-receptor agonist BQ-3020 (10(-8) M) or ET-1 (10(-8) M) plus BQ-788 (10(-7) M). ET-1 concentration-dependently (from 10(-9) to 10(-8)/10(-7) M) stimulated catecholamine release by adrenal slices, and the effect was counteracted by both BQ-123 and BQ-788 (10(-7) M). Collectively, our findings suggest that, when the integrity of adrenal tissue is preserved, a two-fold mechanism underlies the aldosterone secretagogue action of ET-1 in the rat: i) a direct mechanism mediated by ETB receptors located on ZG cells; and ii) an indirect mechanism involving the ETA and ETB receptor-mediated local release of catecholamines, which in turn stimulate ZG cells in a paracrine manner.     

Bosentan, an endothelin receptor antagonist, ameliorates collagen-induced arthritis: the role of TNF-α in the induction of endothelin system genes

Objective

Endothelins (ETs) are involved in several inflammatory events. The present study investigated the efficacy of bosentan, a dual ETA/ETB receptor antagonist, in collagen-induced arthritis (CIA) in mice.

Treatment

CIA was induced in DBA/1J mice. Arthritic mice were treated with bosentan (100?mg/kg) once a day, starting from the day when arthritis was clinically detectable.

Methods

CIA progression was assessed by measurements of visual clinical score, paw swelling and hypernociception. Histological changes, neutrophil infiltration and pro-inflammatory cytokines were evaluated in the joints. Gene expression in the lymph nodes of arthritic mice was evaluated by microarray technology. PreproET-1 mRNA expression in the lymph nodes of mice and in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time PCR. The differences were evaluated by one-way ANOVA or Student’s t test.

Results

Oral treatment with bosentan markedly ameliorated the clinical aspects of CIA (visual clinical score, paw swelling and hyperalgesia). Bosentan treatment also reduced joint damage, leukocyte infiltration and pro-inflammatory cytokine levels (IL-1β, TNFα and IL-17) in the joint tissues. Changes in gene expression in the lymph nodes of arthritic mice returned to the levels of the control mice after bosentan treatment. PreproET mRNA expression increased in PBMCs from rheumatoid arthritis (RA) patients but returned to basal level in PBMCs from patients under anti-TNF therapy. In-vitro treatment of PBMCs with TNFα upregulated ET system genes.

Conclusion

These findings indicate that ET receptor antagonists, such as bosentan, might be useful in controlling RA. Moreover, it seems that ET mediation of arthritis is triggered by TNFα.     

A novel bioactive 31-amino acid ET-1 peptide stimulates eosinophil recruitment and increases the levels of eotaxin and IL-5

OBJECTIVE AND DESIGN: Investigation of the role of a novel inflammatory mediator 31-amino acid endothelin-1 [ET-1 (1-31)], a major ET derivative in granulocytes, in eosinophil recruitment after its subcutaneous administration to mice. METHODS: Various ET-1 derivatives (100 pmol), with or without ET receptor antagonists (200 pmol), were administered subcutaneously to mice, and then the eosinophil migration into and chemokine levels in the injected loci were analyzed. RESULTS: ET-1 (1-31) and a 21-amino acid endothelin-1 (ET-1), but not big ET-1, induced eosinophil migration into the injected loci with a peak after administration for 12 h, and increased the levels of eotaxin and interleukin-5 with peaks at 6 and 24 h, respectively. These effects of ET-1(1-31) and ET-1 were significantly inhibited by an ETA receptor antagonist, BQ-123, but not by an ETB receptor antagonist, BQ-788. CONCLUSION: Novel bioactive ET-1 (1-31) induces local eosinophil migration, and increases in eotaxin and interleukin-5 through an ETA or ETA-like receptor.     

Anti-inflammatory effect of quinoline alkaloid skimmianine isolated from Ruta graveolens L.

Objective

The present study evaluates the anti-inflammatory effect of the quinoline alkaloid skimmianine (SKM), isolated from Ruta graveolens L., against carrageenan-induced acute inflammation.

Methods

SKM at a dose of 5.0 mg/kg body weight was found to be the minimal concentration for maximal edema inhibition. Carrageenan suspension was administered into the sub-plantar tissue of the right hind paw 1 h after SKM and diclofenac (20 mg/kg) administration (i.p.). Paw edema was determined 3 h after carrageenan administration. The rats were then killed and mRNA expressions of TNF-α and IL-6, levels of PGE₂ and TBARS, activities of COX-2, 5-LOX, SOD, catalase, glutathione peroxidase (GPx) and myeloperoxidase (MPO) and the level of nitrite were measured.

Results

SKM treatment resulted in a decrease in the mRNA levels of TNF-α and IL-6, which are upstream events of the inflammatory cascade. The levels of PGE₂ and NO and the activities of COX-2 and 5-LOX were also significantly reduced after SKM treatment. Neutrophil infiltration, lipid peroxidation and associated oxidative stress in the paw tissue were reduced following SKM treatment.

Conclusion

These results support the anti-inflammatory properties of skimmianine and its multi-targeted mechanism of action, suggesting its potential therapeutic efficacy in various inflammatory diseases.     

Anti-inflammatory and anti-arthritic activities of 3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione (β-lapachone)

Objective and design

The purpose of this study was to evaluate the anti-inflammatory and anti-arthritic activities of 3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione (β-lapachone; β-lap) and to elucidate its probable mode of action.

Methods

Carrageenan-induced paw edema, cell migration evaluation and production of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-6 and nitric oxide were used for this study. Freund’s complete adjuvant (FCA)-induced arthritis was used as a model of chronic inflammation. β-Lap was tested in doses of 40 and 60 mg/kg, orally.

Results

In the paw edema test, the dose of 60 mg/kg gave a higher percentage inhibition of edema (49.3 %) than control. β-Lap inhibited neutrophil migration and reduced concentrations of TNF-α, IL-6 and NO in peritoneal exudates of animals with peritonitis. In the arthritis test, β-lap inhibited edema and NO production in the serum of treated animals.

Conclusion

Significant anti-inflammatory and anti-arthritic activities were observed in animals treated with β-lap. The effects of β-lap can be attributed in part to immunomodulation with reduction of pro-inflammatory cytokines and NO.     

Colitis generates remote antinociception in rats: the role of the l-arginine/NO/cGMP/PKG/KATP pathway and involvement of cannabinoid and opioid systems

Objective and design

The aim of this study was to investigate the possible involvement of the NO/cGMP/PKG/K _ATP ⁺ pathway, cannabinoids and opioids in remote antinociception associated with 2,4,6-trinitrobenzene sulph onic acid (TNBS)-induced colitis.

Methods

TNBS-induced colitis was induced by intracolonic administration of 20 mg of TNBS in 50 % ethanol. After induction, carrageenan (500 μg/paw) or prostaglandin (PG) E₂ (100 ng/paw) was injected in the rat’s plantar surface and hypersensitivity was evaluated by the electronic von Frey test. Rats were pre-treated with l-Noarg one hour before carrageenan injection. l-Arginine was given 10 min before l-Noarg injections. ODQ, KT 5823, glibenclamide (Glib), naloxone and AM 251 or AM 630 were administered 30 min prior to carrageenan or PGE₂ treatments.

Results

Colitis induction by TNBS reduced PGE₂ or carrageenan-induced hypersensitivity. Antinociception produced by TNBS-induced colitis was reversed significantly (P < 0.05) by l-Noarg, ODQ, KT 5823, glibenclamide, naloxone, AM251 and AM630 treatments.

Conclusions

TNBS-induced colitis causes antinociception in the rat paw. This disorder appears to be mediated by activation of the NO/cGMP/PKG/K_ATP pathway, endocannabinoids and endogenous opioids. This information may contribute to a better understanding of peripheral neurological dysfunctions occurring in Crohn’s disease.     

Endothelins modulate inflammatory reaction in zymosan-induced arthritis: participation of LTB4, TNF-alpha, and CXCL-1

Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. ET-1 levels are increased in plasma and synovial membrane of rheumatoid arthritis (RA) patients, but the evidence that ETs participate in RA physiopathology is limited. The present study investigated the involvement of ETs in neutrophil accumulation and edema formation in the murine model of zymosan-induced arthritis. Intra-articular (i.a.) administration of selective ET(A) or ET(B) receptor antagonists (BQ-123 and BQ-788, respectively; 15 pmol/cavity) prior to i.a. zymosan injection (500 mug/cavity) markedly reduced knee-joint edema formation and neutrophil influx to the synovial cavity 6 h and 24 h after stimulation. Histological analysis showed that ET(A) or ET(B) receptor blockade suppressed zymosan-induced neutrophil accumulation in articular tissue at 6 h. Likewise, dual blockade of ET(A)/ET(B) with bosentan (10 mg/kg, i.v.) also reduced edema formation and neutrophil counts 6 h after zymosan stimulation. Pretreatment with BQ-123 or BQ-788 (i.a.; 15 pmol/cavity) also decreased zymosan-induced TNF-alpha production within 6 h, keratinocyte-derived chemokine/CXCL1 production within 24 h, and leukotriene B(4) at both time-points. Consistent with the demonstration that ET receptor antagonists inhibit zymosan-induced inflammation, i.a. injection of ET-1 (1-30 pmol/cavity) or sarafotoxin S6c (0.1-30 pmol/cavity) also triggered edema formation and neutrophil accumulation within 6 h. Moreover, knee-joint synovial tissue expressed ET(A) and ET(B) receptors. These findings suggest that endogenous ETs contribute to knee-joint inflammation, acting through ET(A) and ET(B) receptors and modulating edema formation, neutrophil recruitment, and production of inflammatory mediators.     

Endothelin synthesis and receptors in porcine kidney

As insufficient information on the endothelin (ET) system in the porcine kidney is available at present, we investigated renal ET-1 synthesis and ET receptors in this species. Because ET specifically affects renal and glomerular haemodynamics and distal tubular reabsorption, we studied ET-1 synthesis in isolated glomeruli and in inner medullary collecting duct (IMCD) cells and preproET-1 mRNA in renal cortex, isolated glomeruli and papillary tissue. In addition, we characterized density and properties of ET receptors in membranes from isolated glomeruli and papillary tissue. In contrast to isolated IMCD cells, which synthesized 120 +/- 11 fmol h(-1) mg-1 protein of ET-1, no such synthesis was found with isolated glomeruli in our assay system. Nevertheless, with RT-PCR preproET(-1) mRNA was clearly present in renal cortex and glomeruli as well as in papillary tissue. Glomerular membranes were found to have ET receptors with Bmax of 1.6 +/- 0.2 pmol mg-1 protein and Kd of 311 +/- 33 pmol L(-1). Using BQ-123 (10-5 M), a specific blocker of ETA receptors, we found that 58% of total receptors are ETA receptors. Thus, presumably 42% are ETB receptors (Bmax 0.7 +/- 0.1 pmol mg-1 protein; Kd 429 +/- 110 pmol L(-1)). Bosentan (10-5 M), an ETA- and ETB-receptor antagonist, blocked all ET receptors in glomerular membranes. Papillary membranes showed ET receptors with Bmax of 2.1 +/- 0.2 pmol mg-1 protein and Kd of 137 +/- 11 pmol L(-1). In the presence of BQ-123 (10-5 M) we found that all receptors are ETB receptors (Bmax 2.3 +/- 0.4 pmol mg-1 protein; Kd 162 +/- 25 pmol L(-1)). Bosentan (10-5 M) again blocked all ET receptors in papillary membranes, thus confirming our previous finding that IMCD cells possess high-affinity ETB receptors mediating the diuretic effects of ET. Thus, in the porcine kidney the ET system may act in an autocrine/paracrine manner at the glomerular as well as at the IMCD level.     

Swertiamarin ameliorates inflammation and osteoclastogenesis intermediates in IL-1β induced rat fibroblast-like synoviocytes

Objective and design

Rheumatoid arthritis is a chronic inflammatory and autoimmune disease that leads to aggressive joint cartilage and bone destruction. Swertiamarin is a secoiridoid glycoside found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in the Indian system of traditional medicine. In the present study, the potential of swertiamarin was evaluated in IL-1β induced fibroblast-like synoviocytes (FLS).

Methods

The FLS were isolated from Freund’s Complete Adjuvant induced arthritic (AA) rats and cultured with IL-1β. The normal FLS and AA-FLS were cultured and used for subsequent experiment in fibroblastic morphology form. The efficacy of swertiamarin (10–50 μg/ml) was evaluated on mRNA and protein expression levels of inflammatory and osteoclastogenesis mediators. The efficacy was also evaluated on p38 MAPKα levels with time course studies (2, 4, 6, 8 and 12 h).

Results

IL-1β induced cell proliferation (149.46 ± 13.73 %) and NO production (162.03 ± 11.03 %) in AA-FLS; treatment with swertiamarin controlled proliferation (82.77 ± 4.22 %) and NO production (82.06 ± 3.91 % at 50 μg/ml) in a dose-dependent manner. It also significantly (P < 0.05) modulated the expression of apoptotic marker (caspase 3), proinflammation mediators (TNFα, IL-6, PGE2, COX-2, iNOS, MMPs) and osteoclastogenic mediator (RANKL) at both the mRNA and protein levels. Treatment with swertiamarin inhibited the levels of p38 MAPKα in a dose-dependent manner and also significantly (P < 0.05) attenuated the release of the same in time dependent mode.

Conclusion

These findings suggest that treatment with swertiamarin attenuated IL-1β induced FLS, and it revealed anti-inflammatory potential by attenuating aggressive FLS.     

Acute hyperglycaemia does not alter nitric oxide-mediated microvascular function in the skin of adolescents with type 1 diabetes

Purpose

We assessed the impact of an acute bout of hyperglycaemia on nitric oxide (NO)-mediated microvascular function in the skin of adolescents with type 1 diabetes (T1DM).

Methods

Twelve subjects (12–18 years) with T1DM were randomised into a control (n = 6) or hyperglycaemia (n = 6) group. Hyperinsulinaemic clamps were used to manipulate blood glucose level (BGL). Following a baseline period, where all subjects were euglycaemic (20 min), the experimental phase began. During the experimental phase, BGL was elevated to 16.7 ± 0.9 mmol L^?1 in the hyperglyceamic group, while it was maintained at euglycaemia (5.5 ± 0.1 mmol L^?1) in the control group. Simultaneously, cutaneous microvascular function (% max cutaneous vascular conductance, CVC%) was assessed using laser Doppler fluxometry following stimulation of skin blood flow using localised heating (42 °C). To determine the NO contribution to skin blood flow, two microdialysis sites were assessed, one perfused with Ringers and the other with the NO blocker, NG-monomethyl-l-arginine (l-NMMA).

Results

In the hyperglycaemic group, acute increase in BGL was not associated with changes in skin blood flow (CVC% 82.4 ± 8.7 % at 5.5 ± 0.1 mmol L^?1 vs 79.5 ± 9.1 % at 16.7 ± 0.9 mmol L^?1, unpaired t tests, P = 0.588) or the contribution of NO to vasodilation.

Conclusions

These results suggest that, in our group of adolescents with type 1 diabetes, acute hyperglycaemia did not affect skin microvascular NO-mediated function.     

Inflammatory mediators of systemic inflammation in neonatal sepsis

Objective and design

Sepsis refers to severe systemic inflammation in response to invading pathogens. To understand the molecular events that initiate the systemic inflammatory response, various inflammatory mediators were analyzed in neonatal sepsis samples and compared with normal samples.

Materials and methods

We initially measured the levels of the various classical inflammatory mediators such as acute phase proteins [C-reactive protein (CRP) and procalcitonin (PCT)], granule-associated mediators (NE, MPO and NO), proinflammatory cytokines [tumour necrosis factor-α (TNFα), IL-1β and IL-6), antiinflammatory cytokines (IL-10 and IL-13) and chemokines [IL-8 and monocyte chemotactic protein (MCP-1)] and novel cytokines (IL-12/IL-23p40, IL-21 and IL-23) using ELISA. We also used the human inflammation antibody array membrane to profile the inflammatory proteins that are involved in neonatal sepsis.

Results

There were significantly higher levels of CRP (5.4 ± 0.70 mg/L), PCT (1.500 ± 0.2400 μg/L); NE (499.2 ± 22.01 μg/L), NO (54.22 ± 3.131 μM/L); TNFα (396.6 ± 37.40 pg/mL), IL-1β (445.3 ± 34.25 pg/mL), IL-6 (320.9 ± 43.38 pg/mL); IL-8 (429.5 ± 64.08 pg/mL) MCP-1 (626.25 ± 88.91 pg/mL), IL-10 (81.80 ± 9.45 pg/mL), IL-12/IL-23p40 (30.25 ± 0.6 pg/mL), IL-21 (8,263.3 ± 526.8 pg/mL) and IL-23 (6,083 ± 781.3 pg/mL) in neonates with sepsis compared to normal. The levels of MPO (21.20 ± 3.099 ng/mL) were downregulated, whereas there was no change in IL-13 (188.7 ± 10.63 pg/mL) levels in septic neonates when compared with normal. Using the human inflammation antibody array membrane, we detected the presence of 17 inflammatory proteins such as IL-3, IL6R, IL12p40, IL-16, TNFα, TNFβ, TNF R1, chemokines I-309, IP-10 (IFN-γ inducible protein 10), MCP-1, MCP-2, MIP 1β (macrophage inflammatory protein), MIP-1δ, eotaxin-2, growth factors TGFβ1 (transforming growth factor beta), PDGF (platelet derived growth factor), and cell adhesion molecule ICAM-1 (intracellular adhesion molecule) that were upregulated whereas RANTES which was downregulated in neonatal sepsis.

Conclusion

The simultaneous secretion and release of multiple mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors were found to be involved in the initiation of systemic inflammation in neonatal sepsis. Therefore, measuring the concentration of multiple mediators may help in the early detection of neonatal sepsis and help to avoid unnecessary antibiotic treatment.     

Inflammatory Models

Objective and design

The sigma 1 (σ₁) receptor, which is widely distributed in the CNS in areas that are known to be important for pain control, may play a role in persistent pain characterized by the hypersensitivity of nociceptive transmission. Here, we investigated the effect of σ₁ blockade in an inflammatory pain model.

Treatment and methods

An intraplantar injection of carrageenan (2 %) was used to induce paw inflammation. The effects of the σ₁ antagonist (+)-MR200, given subcutaneously at a dose of 0.1, 0.25, 0.5,1, 1.5, and 2 mg/kg prior to injection of carrageenan, on inflammatory pain and inflammation were assessed. Mechanical allodynia with von Frey filaments, thermal hyperalgesia with the plantar test and edema evaluation with a plethysmometer were measured. Intergroup comparisons were assessed by one- or two-way analysis of variance when appropriate, followed by post-hoc tests (Dunnett’s test for one-way or Bonferroni for two-way ANOVA).

Results

(+)-MR200 dose-dependently prevented allodynia and hyperalgesia induced by carrageenan. Furthermore, it reduced paw edema with a significant inhibition percentage of 37.82 % at 3 h after carrageenan treatment.

Conclusions

The blockade of the σ₁ receptor with the selective antagonist (+)-MR200 may contribute to the suppression of the typical symptoms of inflammatory pain.     

Fetal natural killer cell function is suppressed.

Endothelins (ETs), potent vasoconstricting peptides, are produced by macrophages upon stimulation and may participate in the amplification or regulation of the inflammatory response. However, it is not clear whether ETs can act in an autocrine manner on macrophages and which role they play in relationship with other cytokines. To address these issues, we studied the effects of ETs on the production of inflammatory cytokines by mouse peritoneal macrophages or by a retrovirus-transformed microglial cell line. Here, we report that ET-2, but not ET-1 or ET-3, is able to stimulate the production of interleukin-1 (IL-1) and interleukin-6 (IL-6) by peptone-elicited mouse macrophages (pMO). In contrast, ET-3 and ET-1, but not ET-2, are active on microglial cells. No tumour necrosis factor-alpha (TNF-alpha) or nitric oxide (NO) were detected in the supernatants of ET-stimulated cultures. The activity of ET-2 on pMO was time and dose dependent and was inhibited by the addition of ETA and ETB receptor antagonists, BQ123 and IRL1038, respectively. In addition, when pMO were stimulated by interferon-gamma (IFN-gamma) in the presence of ET-2, a significant inhibition of IL-6 and IL-1 production was observed compared with the effects of the same doses of IFN-gamma or ET-2 used separately. The inhibition was specifically due to the activity of ET-2, since it was reversed by the addition of BQ123 or IRL1038. Similar results were seen when the content of NO in the supernatants of pMO stimulated by IFN-gamma plus ET-2 was evaluated. These results suggest that ETs may possess both a pro-inflammatory action on macrophages from different tissues and a regulatory activity on IFN-gamma.     

Central and peripheral anti-inflammatory effects of maprotiline on carrageenan-induced paw edema in rats

Objective

To explore the site of action of maprotiline, as an atypical antidepressant, on carrageenan-induced paw edema.

Subjects

Male Wistar rats were used.

Methods

Firstly, the anti-inflammatory effect of systemic maprotiline (12.5, 25 and 50 mg kg^?1) was assessed using a paw edema model. Secondly, different doses of maprotiline were administrated intracerebroventricularly, intrathecally and locally before carrageenan challenge. Finally, we tried to reverse the anti-inflammatory effect of maprotiline by propranolol (10 mg kg^?1), prazosin (4 mg kg^?1), yohimbine (10 mg kg^?1), naloxone (4 mg kg^?1) and mifepristone (5 mg kg^?1).

Results

Systemic, intracerebroventricular and subplantar application of maprotiline significantly inhibited peripheral edema, but intrathecal maprotiline did not alter the degree of paw swelling. The applied antagonists failed to change the anti-inflammatory activity of maprotiline.

Conclusion

These results demonstrate that maprotiline has a potent anti-inflammatory effect and this effect is linked to the peripheral and supraspinal actions of the drug.     

Preclinical evaluation of the urokinase receptor-derived peptide UPARANT as an anti-inflammatory drug

Background

Inflammation plays a key role in the pathogenesis of several chronic diseases. The urokinase plasminogen activator receptor (uPAR) exerts a plethora of functions in both physiological and pathological processes, including inflammation.

Objective and design

In this study, we evaluated the anti-inflammatory effect of a novel peptide ligand of uPAR, UPARANT, in different animal models of inflammation.

Subjects and treatment

Rats and mice were divided in different groups (n = 5) for single or repeated administration of vehicle (9% DMSO in 0.9% NaCl), UPARANT (6, 12 and 24 mg/kg) or dexamethasone (2 mg/kg). Animals were subjected to carrageenan-induced paw oedema or zymosan-induced peritonitis.

Methods

UPARANT effects were tested on: (1) the carrageenan-induced paw oedema volume, (2) the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the nitrite/nitrate (NOx) levels in the paw exudates, (3) cells recruitment into the peritoneal cavity after zymosan injection and (4) NOx levels in the peritoneal lavage.

Results

UPARANT (12 and 24 mg/kg) reduced inflammation in both experimental paradigms. Analysis of pro-inflammatory enzymes revealed that administration of UPARANT reduced iNOS, COX2 and NO over-production.

Conclusions

Our study provides a solid evidence that UPARANT reduces the severity of inflammation in diverse animal models, thus representing a novel anti-inflammatory drug with potential advantages with respect to the typical steroidal agents.

     

Impact of chlorhexidine mouth rinse use on postextraction infection via nitric oxide pathway

Objective and design

Nitric oxide (NO) has been linked to inflammatory reactions, tissue destruction, host defense, and wound healing in oral diseases. It is known that arginase enzyme controls the synthesis of NO through arginine depletion. This study evaluated the arginase–NO pathway alteration in response to tissue injury after dental extraction surgery and the effect of postoperative use of 0.2% chlorhexidine gluconate rinse (CHX).

Materials and methods

This study included 28 individuals who had impacted mandibular third molars. They were randomly divided into two groups. Group A was comprised of 13 individuals who used postoperative CHX (0.2%) rinse, while group B included 15 individuals who did not use postoperative CHX rinse. For each patient, periodontal inflammatory status was evaluated. Salivary and gingival tissue samples were obtained before and 1 h and 1 week after the surgery to determine the NO level and arginase activity using spectrophotometric methods.

Results

NO level of tissue samples displayed an insignificant decrease in both groups postoperatively. However, arginase activity of tissue samples was significantly higher in group B compared to group A 1 week after surgery (p ≤ 0.01). There were no significant changes in salivary arginase activity and NO level in both groups before or after the third molar surgery.

Conclusion

Results indicated that good oral hygiene following periodontal treatment prior to third molar surgery was related to an increase in arginase activity, which may improve wound healing. However, use of CHX rinse following periodontal treatment had no pronounced effect on the arginase–NO pathway.     

Augmented expression of endothelin-1, endothelin-3 and the endothelin-B receptor in breast carcinoma

AIMS: Endothelins (ETs) are peptides expressed in many tumours which may stimulate angiogenesis and desmoplasia. Because ETs have not been extensively studied mammary neoplasia, we assessed ET protein and mRNA expression and receptor mRNA expression in normal and neoplastic breast tissues. METHODS AND RESULTS: Tissues from five normal breasts, six fibroadenomas, seven ductal carcinomas in situ (DCIS) and 25 invasive carcinomas were stained with anti-ET-1 and anti-ET-3 antibodies and analysed using a grading system. ET-1, ET-3, ETA and ETB mRNA expression was assessed by quantitative RT-PCR from eight carcinomas and five normals. Weak staining for ET-1 and ET-3 was detected in all normals. Moderate to strong staining was seen in 72% and 64% of carcinomas for ET-1 and ET-3, respectively. Most fibroadenomas showed weak positivity for ET-1 (83%) and ET-3 (67%). ET-1 and ET-3 mRNA levels were upregulated in carcinomas compared with normal breast. No ETA mRNA was not detected in any tissue. ETB mRNA was detected in normal breast and was increased in carcinomas. CONCLUSION: These results suggest that the ET system is altered in breast carcinomas and this may be of importance in the progression from in-situ to invasive carcinoma.